Succinate nano-drug as well as preparation method and application thereof

Abstract

The invention provides a succinate nano-drug as well as a preparation method and application thereof. The succinate nano-drug is prepared from ammonium succinate and sodium oleate through a microemulsion method. When the succinate nano-drug enters tumor cells, pyroptosis can be triggered, and expression up-regulation of a main histocompatibility complex I (MHC-I) can be stimulated. The occurrence of pyroptosis will release a large number of injury-associated molecular patterns (DAMPs) to trigger immune activation; and up-regulation of MHC-I expression can promote immune presentation of tumor cells. Under the combined action of immune activation induced by pyroptosis and MHC-I expression up-regulation, the succinate nano-drug not only can effectively activate whole-body anti-tumor immune response, but also can effectively increase tumor cell antigen presentation, so that tumor growth and metastasis are efficiently inhibited, and the immune checkpoint blocking treatment effect is improved. The succinate nano-drug is simple to prepare, mild in reaction condition, free of heavy metal elements and high in biological safety, and shows a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drugs, and in particular relates to a succinate nano drug, a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy is a treatment method that fights cancer by activating the body's own immune system. In recent years, immunotherapy represented by immune checkpoint blockade (ICB) therapy has been widely used in clinical practice, showing great prospects and advantages. However, the low immune response rate in existing immunotherapy (generally around 20%) still greatly limits the further development of tumor immunotherapy. Tumor immune escape is one of the main reasons for the failure of cancer immunotherapy. For example, the absence or downregulated expression of MHC-I on the surface of cancer cells can cause patients to be resistant to ICB treatment, thereby avoiding immune killing. MHC-I is located on the cell surface, and its main function is to present antigens to T cells to distinguish between self (self cells and tissues) and non-self (invaded or modified self). When an immune response occurs, CD8 + T cells recognize peptide fragments (called antigens) presented on the MHC-I molecules on the surface of cancer cells and bind to them, thereby killing cancer cells. Therefore, the MHC-I antigen presentation pathway is the key factor that determines the CD8 + T cell specificity and activation is a key mechanism, and it is also an important target for cancer immunotherapy. However, more and more studies have shown that cancer cells can downregulate MHC-I positive regulators or upregulate MHC-I negative regulators through a variety of mechanisms to interfere with the MHC-I antigen presentation pathway and reduce the level of MHC-I expression on the tumor cell surface. This effect greatly reduces the CD8 + The recognition and killing of tumor cells by T cells increases the chance of tumor immune escape, thereby generating resistance to immunotherapy. In short, MHC-I, as an important antigen presenting molecule, plays a vital role in tumor antigen presentation. Targeted upregulation of MHC-I expression may become an effective immune escape inhibition strategy, thus providing a new idea for enhancing clinical tumor immunotherapy.

[0003] Existing studies believe that mutations or deletions of key genes (Traf3, B2M, TAP1 / 2, JAK, etc.) that affect the MHC-I antigen presentation pathway are the main reason for the downregulation of MHC-I antigen presentation in most tumors. Existing technologies mainly increase the expression of MHC-I molecules by gene knockout. For example, research by Professor Liu Xiaole and Professor Myles Brown of Harvard University in the United States showed that knockout of the Traf3 gene can significantly upregulate the expression of MHC-I molecules, thereby significantly improving the effect of immune checkpoint inhibitors on tumor treatment (Cancer Discov., 2021, 11, 1524-1541).

[0004] The existing technology mainly relies on the strategy of increasing the expression of MHC-I molecules through gene knockout, which is prone to off-target toxicity, and the high cost of treatment also limits its further application. Therefore, how to effectively increase the expression of MHC-I in tumor cells and reduce tumor immune escape is still a key scientific problem that is currently facing and urgently needs to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a succinate nano drug, a preparation method and application thereof. The succinate nano drug of the present invention can stimulate the up-regulation of MHC-I expression and improve the ICB treatment effect. The preparation is simple, the reaction conditions are mild, the drug does not contain heavy metal elements, and the biosafety is high, showing good application prospects.

[0006] The invention provides a succinate nano drug, which is prepared from ammonium succinate and sodium oleate by a microemulsion method.

[0007] Preferably, the particle size of the succinate nanomedicine is 10 to 200 nm.

[0008] Preferably, the molar ratio of ammonium succinate to sodium oleate is 1:(1-5).

[0009] The present invention provides a method for preparing the succinate nanomedicine as described above, comprising the following steps:

[0010] Sodium succinate and sodium oleate solutions are mixed and reacted by a microemulsion method to obtain succinate nanomedicine.

[0011] Preferably, the sodium oleate solution comprises sodium oleate and an organic solvent, and the organic solvent comprises a mixture of n-hexane, anhydrous ethanol and oleylamine; the volume ratio of n-hexane, anhydrous ethanol and oleylamine is (5-15):(1-5):1.

[0012] Preferably, the concentration of sodium oleate in the sodium oleate solution is 0.5-2.5 mg / mL.

[0013] Preferably, after dispersing ammonium succinate in water, the dispersion of sodium succinate is mixed with the sodium oleate solution;

[0014] The concentration of ammonium succinate in the ammonium succinate dispersion is 50-500 mg / mL.

[0015] Preferably, the sodium succinate and sodium oleate solutions are mixed under stirring conditions, and the stirring speed is 200-1000 rpm.

[0016] Preferably, the reaction temperature is 15-40° C., and the reaction time is 3-60 min.

[0017] The present invention provides an anti-tumor drug, comprising the succinate nano drug described above or the succinate nano drug prepared by the preparation method described above.

[0018] The present invention provides a succinate nano drug, which is prepared from ammonium succinate and sodium oleate by a microemulsion method. When the succinate nano drug enters the tumor cells, it is rapidly degraded in the acidic lysosomes of the tumor cells to release a large amount of Na + ions and succinate ions, causing a surge in ion concentration and osmotic pressure in tumor cells and the accumulation of succinate, thereby triggering cell pyroptosis and stimulating upregulation of MHC-I expression. The occurrence of cell pyroptosis will release a large number of damage-associated molecular patterns (DAMPs) to trigger immune activation; upregulation of MHC-I expression will promote the immune presentation of tumor cells. Therefore, under the combined effects of immune activation induced by cell pyroptosis and upregulation of MHC-I expression, succinate nanomedicines can not only effectively activate systemic anti-tumor immune responses, but also effectively increase tumor cell antigen presentation, thereby efficiently inhibiting tumor growth and metastasis and improving the therapeutic effect of ICB. The succinate nanomedicine is simple to prepare, has mild reaction conditions, does not contain heavy metal elements, has high biosafety, and shows good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 This is a transmission electron microscope photo of the succinate nanomedicine obtained in Example 1 of the present invention;

[0021] Figure 2 This is the X-ray diffraction pattern of the succinate nanomedicine obtained in Example 1 of the present invention;

[0022] Figure 3 This is a transmission electron microscope photo of the succinate nanomedicine obtained in Example 2 of the present invention;

[0023] Figure 4 This is a transmission electron microscope photo of the succinate nanomedicine obtained in Example 3 of the present invention;

[0024] Figure 5 The data of up-regulation of MHC-I expression in tumor cells stimulated by the succinate nanomedicine obtained in Example 1 of the present invention;

[0025] Figure 6This is a photograph of the cell morphology of mouse breast cancer cells (4T1) treated with the succinate nanomedicine obtained in Example 1 of the present invention;

[0026] Figure 7 The cytotoxicity data of the succinate nanomedicine obtained in Example 1 of the present invention after treating 4T1 cells;

[0027] Figure 8 Figure 1 shows the changes in mouse tumors after treatment with succinate nanomedicine. Figure A shows a photo of mouse tumors, Figure B shows a curve showing the changes in mouse tumor volume over time, and Figure C shows a curve showing the changes in mouse weight over time.

[0028] Fig. 9 Figure 1 shows the expression of different immune cells in mice after treatment with succinate nanomedicine. Figure A shows the maturation of dendritic cells in the spleen of mice, Figure B shows the statistical data of mature dendritic cells in the spleen of mice, and Figure C shows the CD4 + T cell situation, Figure D shows CD4 + T cell statistics, Figure E shows CD8 + T cell status, Figure F shows CD8 + T cell statistics. DETAILED DESCRIPTION

[0029] The invention provides a succinate nano drug, which is prepared from ammonium succinate and sodium oleate by a microemulsion method.

[0030] In the present invention, the succinate nanomedicine is a nanoparticle of sodium succinate, and the particle size is preferably 10 to 200 nm, more preferably 50 to 150 nm.

[0031] In the present invention, the molar ratio of ammonium succinate to sodium oleate is preferably 1:(1-5).

[0032] The present invention also provides a method for preparing the succinate nanomedicine described above, comprising the following steps:

[0033] Sodium succinate and sodium oleate solutions are mixed and reacted by a microemulsion method to obtain succinate nanomedicine.

[0034] The present invention preferably disperses ammonium succinate in water to obtain an ammonium succinate dispersion, then mixes the ammonium succinate dispersion with a sodium oleate solution under stirring, reacts by a microemulsion method, and collects the precipitate by centrifugation after the reaction is completed to obtain a succinate nano drug.

[0035] In the present invention, in the ammonium succinate dispersion, the concentration of ammonium succinate is preferably 50-500 mg / mL, more preferably 100-400 mg / mL, such as 50 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, 500 mg / mL, preferably a range value with any of the above values ​​as the upper or lower limit.

[0036] In the present invention, the sodium oleate solution preferably includes sodium oleate and an organic solvent, and the organic solvent preferably includes a mixture of n-hexane, anhydrous ethanol and oleylamine. The volume ratio of n-hexane, anhydrous ethanol and oleylamine is preferably (5-15):(1-5):1, more preferably (8-12):(2-4):1, and most preferably (9-11):(2-4):1. Specifically, in an embodiment of the present invention, it can be 10:3:1.

[0037] In the present invention, in the sodium oleate solution, the concentration of sodium oleate is preferably 0.5-2.5 mg / mL, more preferably 1-2 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, preferably a range value with any of the above values ​​as the upper or lower limit.

[0038] In the present invention, the molar ratio of ammonium succinate to sodium oleate in the sodium oleate solution is preferably 1:(1-5), more preferably 1:(2-4), such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, preferably a range value with any of the above values ​​as the upper or lower limit.

[0039] In the present invention, the stirring speed during the mixing process is preferably 200 to 1000 rpm, more preferably 300 to 800 rpm.

[0040] In the present invention, the reaction temperature of the microemulsion method is preferably 15 to 40°C, more preferably 20 to 30°C, and most preferably the reaction is carried out at room temperature. The reaction time of the microemulsion method is preferably 3 to 60 min, more preferably 5 to 50 min, such as 3 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, preferably a range value with any of the above values ​​as the upper or lower limit.

[0041] The present invention also provides an anti-tumor drug, comprising the succinate nano drug described above.

[0042] When succinate nanomedicine enters tumor cells, it is rapidly degraded in the acidic lysosomes of tumor cells and releases a large amount of Na + ions and succinate ions, causing a surge in ion concentration and osmotic pressure in tumor cells and the accumulation of succinate, thereby triggering cell pyroptosis and stimulating upregulation of MHC-I expression. The occurrence of cell pyroptosis will release a large number of damage-associated molecular patterns (DAMPs) to trigger immune activation; upregulation of MHC-I expression will promote the immune presentation of tumor cells. Therefore, under the combined effects of immune activation induced by cell pyroptosis and upregulation of MHC-I expression, succinate nanomedicines can not only effectively activate systemic anti-tumor immune responses, but also effectively increase tumor cell antigen presentation, thereby efficiently inhibiting tumor growth and metastasis and improving the therapeutic effect of ICB. The succinate nanomedicine is simple to prepare, has mild reaction conditions, does not contain heavy metal elements, has high biosafety, and shows good application prospects.

[0043] In order to further illustrate the present invention, a succinate nanomedicine provided by the present invention, its preparation method and application are described in detail below in conjunction with examples, but it should not be understood as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 10 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 10 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0046] Transmission electron microscopy was used to characterize the morphology of succinate nanoparticles. Figure 1 As shown in the figure, it can be seen that the succinate nanoparticles prepared by the microemulsion method are spherical particles with uniform size. At the same time, X-ray diffraction was used to characterize the structure of the succinate nanoparticles. The results are shown in Figure 2 As shown, it is proved that it has a pure phase structure of sodium succinate.

[0047] Example 2

[0048] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0049] Example 3

[0050] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 100 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 100 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0051] Example 4

[0052] 7 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0053] Example 5

[0054] 35 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0055] Example 6

[0056] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 3 minutes. The precipitate was collected by centrifugation to obtain the succinate nanodrug.

[0057] Example 8

[0058] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At room temperature, under the condition of a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at room temperature of 25°C for 60 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0059] Example 9

[0060] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At 28°C and a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at 28°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0061] Example 10

[0062] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At 15°C and a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at 15°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0063] Embodiment 11

[0064] 20 mg of sodium oleate was dispersed in a mixed solution containing 10 ml of n-hexane, 3 ml of ethanol and 1 ml of oleylamine, and 5 mg of ammonium succinate was dispersed in 50 μl of deionized water. At 40°C and a stirring rate of 1000 rpm, 50 μl of ammonium succinate aqueous solution was added dropwise to the sodium oleate mixed solution, and the reaction was continued at 40°C for 10 minutes. The precipitate was collected by centrifugation to obtain the succinate nanomedicine.

[0065] The present invention detects MHC-I on the surface of mouse tumor cells induced by the succinate nanomedicine obtained in Example 1. The tumor of the mouse after treatment was prepared into a single cell suspension, and then incubated with a fluorescently labeled MHC-I antibody. After the incubation, it was detected by flow cytometry. The detection results are as follows Figure 5 As shown, it is demonstrated that the synthesized succinate nanomedicine can stimulate the upregulation of MHC-I expression in tumor cells.

[0066] The present invention conducted an in vitro cell pyroptosis detection experiment on the succinate nanodrug obtained in Example 1. The test method is as follows: 4T1 cells were seeded in a 12-well plate at a density of 100,000 cells per well. After culturing in RMPI medium for 12 hours, 200 micrograms per milliliter of succinate nanodrug was added and incubated for 12 hours. The cell morphology was photographed using an inverted fluorescence microscope. The test results are shown in FIG. Figure 6 As shown, cells treated with succinate nanomedicine showed obvious cell blebbing, proving the occurrence of cell pyroptosis.

[0067] The present invention performs an in vitro cytotoxicity analysis test on the succinate nanodrug obtained in Example 1. The test method is as follows: the cytotoxicity is determined by the MTT method. 4T1 cells are seeded in a 96-well plate at a density of 6000 cells per well. After culturing in RMPI medium for 12 hours, different concentrations of succinate nanodrugs (concentration range of 0-200 μg mL -1 ) were incubated for 24 hours, and cell proliferation was detected by MTT assay. The test results were as follows Figure 7 As shown, it is shown that the prepared succinate nanomedicine can effectively kill tumor cells.

[0068] The present invention tested the immune activation effect and tumor inhibition effect of the succinate nanodrug obtained in Example 1. BALB / c mice bearing 4T1 tumors were randomly divided into three groups (6 mice in each group), and the succinate nanodrug was injected into the tumor twice on the first day and the third day (the succinate nanodrug was dispersed in PBS solution, and the administration concentration was 40.0 mg Na + (kg BW) -1 Succinate nanoparticles (prepared in Example 1, corresponding to Figure 8 and Fig. 9 C 4 H 4 Na 2 O 4 NPs), succinate small molecule reagents (small molecules free of sodium succinate, corresponding to Figure 8 and Fig. 9 C 4 H 4 Na 2 O 4 )(40.0mg Na + (kg BW) -1 ) or PBS (corresponding to Figure 8 and Fig. 9 During the treatment period, the length (L) and width (W) of each tumor were measured with a caliper. The tumor volume (V) was calculated by the following formula.

[0069]

[0070] In addition, body weight data were collected. At the end of treatment, mice were sacrificed to remove major organs and tumors for further use. According to standard procedures, we obtained single cell suspensions of spleen by digestion. Then, single cell suspensions of spleen were detected by flow cytometry (guava easyCyteTM) using fluorescein-labeled anti-mouse antibodies (FITC anti-mouse CD11c, APC anti-mouse CD86, PE anti-mouse CD80) to look for immature DCs. In addition, single cell suspensions of tumors were also obtained, and T cells (FITC anti-mouse CD4, PE anti-mouse CD3, APC anti-mouse CD8a, 7-ADD) were detected using fluorescein-labeled anti-mouse antibodies. The test results are shown in Figure 8 , Fig. 9 As shown, succinate nanomedicine can effectively activate the body's immune response and effectively inhibit tumor growth. In addition, the weight of mice did not decrease significantly, showing good biosafety.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A succinate nano drug prepared from ammonium succinate and sodium oleate by a microemulsion method.

2. The succinate nano drug according to claim 1, characterized in that The particle size of the succinate nano drug is 10 to 200 nm.

3. The succinate nano drug according to claim 1, characterized in that The molar ratio of the ammonium succinate to the sodium oleate is 1:(1-5).

4. The method for preparing the succinate nano drug according to claim 1, comprising the following steps: Sodium succinate and sodium oleate solutions are mixed and reacted by a microemulsion method to obtain succinate nanomedicine.

5. The preparation method according to claim 4, characterized in that: The sodium oleate solution comprises sodium oleate and an organic solvent, wherein the organic solvent comprises a mixture of n-hexane, anhydrous ethanol and oleylamine; the volume ratio of the n-hexane, anhydrous ethanol and oleylamine is (5-15):(1-5):

1.

6. The preparation method according to claim 4, characterized in that: The concentration of sodium oleate in the sodium oleate solution is 0.5-2.5 mg / mL.

7. The preparation method according to claim 4, characterized in that: After dispersing ammonium succinate in water, the dispersion of sodium succinate is mixed with the sodium oleate solution; The concentration of ammonium succinate in the ammonium succinate dispersion is 50-500 mg / mL.

8. The preparation method according to claim 4, characterized in that: The sodium succinate and sodium oleate solutions are mixed under stirring conditions, and the stirring speed is 200-1000 rpm.

9. The preparation method according to claim 4, characterized in that: The reaction temperature is 15-40° C., and the reaction time is 3-60 min.

10. An anti-tumor drug, characterized in that: The invention comprises the succinate nano drug according to any one of claims 1 to 3 or the succinate nano drug prepared by the preparation method according to any one of claims 4 to 9.

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